Possible posttranslational modification,and its genetic control,in flax genotroph isozymes

Environmentally induced flax genotrophs L and S show heritable shifts in the relative mobilities of peroxidase, esterase, and acid phosphatase isozymes, plus a number of nonspecific glycoproteins. All L isozymes migrated faster than corresponding S isozymes in 10% acrylamide gels. Various aspects of these shifts are reviewed here; it is proposed that posttranslational modification, probably of the carbohydrate moieties of these glycoproteins, underlies the shifts. This proposal is discussed in relation to the switch model for genotroph induction.The financial assistance of the Natural Sciences and Engineering Research Council of Canada is acknowledged with thanks.     

Malate dehydrogenase isozymes in flax genotroph leaves: Differences in apparent molecular weight and charge between and within L and S

Ferguson plots demonstrated that corresponding malate dehydrogenase (MDH) isozymes of Durrant's L and S flax genotrophs differ in apparent molecular weight (MW) and also in net negative charge. The MW differences explain heritable differences in electrophoretic relative mobility (R _m) between corresponding L and S isozymes. The MW for each MDH isozyme was higher for L than for S and resulted in a slowerR _m for L. The net negative charge for each isozyme was higher for L than for S. MDH isozymes also differ in MW within L and S. MW was lower for isozymes in leaves from the bottom of the stem than in leaves from the top of the stem, particularly in L. Integration of information on the MDH isozyme system in the flax genotrophs and information on the peroxidase system suggests the possibility that common modifier loci may controlR _m in both enzymes.The financial assistance of the Natural Sciences and Engineering Research Council of Canada is acknowledged with thanks.     

线粒体DNA与DNA甲基化的关系

线粒体DNA（mitochondrial DNA,mtDNA）遗传信息量虽小,却控制着线粒体一些最基本的性质,对细胞及其功能有着重要影响。mtDNA的损伤与衰老、肿瘤等疾病的发生有关。DNA甲基化是调节基因表达的重要方式之一。mtDNA基因的表达受核DNA（nuclear DNA,nDNA）的调控,mtDNA和nDNA协同作用参与机体代谢调节和发病。本文就近年来mtDNA与DNA甲基化的关系作一综述。     

Histophotometric DNA measurements onUmbelliferae,Solanaceae andCompositae,before and after crown gall tumour induction

Summary Histophotometric measurements of the relative amount of DNA per nucleus in control cells (meristematic cells of the plumula) and in differentiated cells (procambium and parenchyma cells of the first or second internode or leaf mesophyll cells) reveal that the investigated species, belonging to theUmbelliferae, Solanaceae andCompositae, differentiate within the diploid conditions. After crown gall transformation, the nuclear DNA amount remains within the diploid limits. However, a limited number of cells, grouped or spread in the tumour goes through an endomitotic cell cycle and becomes octaploid or even 16-ploid. The conclusion seems justified that a strong relationship exists between the nuclear condition in the host plant and in the tumour tissues. The importance of primary (releated to the mitotic stimulus and dedifferentiation of existing cells at the onset of tumour growth) and secondary phenomena (resulting from the specific physiology in the tumours) is discussed.     

The methylation landscape of tumour metastasis

The metastatic cascade which leads to the death of cancer patients results from a multi‐step process of tumour progression caused by genetic and epigenetic alterations in key regulatory molecules. It is, therefore, crucial to improve our understanding of the regulation of genes controlling the metastatic process to identify predictive biomarkers and to develop more effective therapies to treat advanced disease. The study of epigenetic mechanisms of gene regulation offers a novel approach for innovative diagnosis and treatment of cancer patients. Recent discoveries provide compelling evidence that the methylation landscape (changes in both DNA methylation and histone post‐translational modifications) is profoundly altered in cancer cells and contributes to the altered expression of genes regulating tumour phenotypes. However, the impact of methylation events specifically on the advanced metastatic process is poorly understood compared with the initial oncogenic events. Moreover, the characterisation of a large number of histone‐modifying enzymes has revealed their active roles in cancer progression, via the regulation of specific target genes controlling different metastatic phenotypes. Here, we discuss two main methylating events (DNA methylation and histone‐tail methylation) involved in oncogenesis and metastasis formation. The potential reversibility of these molecular events makes them promising biomarkers of metastatic potential and potential therapeutic targets.     

A comparative analysis of developmental profiles for DNA methylation in 5-azacytidine-induced early-flowering flax lines and their control

Earlier experiments demonstrated that DNA from young plants of 5-azacytidine-induced flax (Linum usitatissimum) lines that flower earlier-than-normal is hypomethylated relative to DNA from their control lines and detected differences in methylation level between plants sampled at different ages, which suggested that the methylation level in flax changes during development. To investigate this possibility, and its potential impact on the difference in methylation level between early-flowering and control lines, developmental profiles were established for the cytosine methylation levels in DNA from post-germination seedlings and from the shoot tips of main stems and the cotyledons sampled throughout vegetative phase. The methylation profiles for two early-flowering lines and their control lines were compared. The methylation profiles were then compared to profiles for DNA content, tissue weight and chlorophyll content (green tissues); these additional parameters provided information on tissue status in terms of cell division, tissue expansion and/or photosynthetic maturity. With one exception, methylation levels were either static or increased with plant age and/or tissue maturity; the highest methylation levels were seen in senescent cotyledons. Although DNA from immature plants or tissues of the early-flowering lines was usually hypomethylated, the hypomethylation was not always apparent in tissues from older plants.     

DNA甲基化与衰老研究进展

DNA甲基化与衰老的研究是近年来生命科学领域研究的热点之一。综述了DNA甲基化理论研究进展和探讨影响甲基化与衰老的主要因素,以揭示两者之间可能存在的联系。     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma

Mature microRNAs (miRNAs) are a class of small non-coding RNAs involved in posttranslational gene silencing. Previous studies found that downregulation of miRNAs is a common feature observed in solid tumors, including hepatocellular carcinoma (HCC). We employed a genome-wide approach to test the hypothesis that DNA methylation alterations in miRNA host genes may cause deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Infinium HumanMethylation27 DNA Analysis BeadChips that include 254 CpG sites covering 110 miRNAs from 64 host genes. Expression levels of three identified miRNAs (miR-10a, miR-10b and miR-196b) were measured in a subset of 37 HCC tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites from 27 host genes significantly differed in DNA methylation levels between tumor and adjacent non-tumor tissues with 53 sites significantly hypermethylated in tumor tissues. Among the 54 significant CpG sites, 15 sites had more than 2-fold tumor/non-tumor changes, 17 sites had differences > 10%, and 10 sites had both features [including 8 significantly hypermethylated CpG sites in the host genes of miR-10a, miR-10b and miR-196b (HOXB4, HOXD4 and HOXA9, respectively)]. Significant downregulation of miR-10a was observed in tumor compared with non-tumor tissues (0.50 vs. 1.73, p = 0.031). The concordance for HOXB4 methylation alteration and dysregulation of miR-10a was 73.5%. No significant change was observed for miR-10b expression. Unexpectedly, miR-196b was significantly upregulated in tumor compared with non-tumor tissues (p = 0.0001). These data suggest that aberrant DNA methylation may lead to dysregulation of miR-10a in HCC tumor tissues.     

DNA methylation and epigenetic defects in carcinogenesis

It is frequently assumed that DNA-damaging agents are carcinogenic because they induce mutations. However, another strong possibility is that the damage leads to heritable changes in the methylation of cytosine in DNA. Considerable evidence exists that gene expression in mammalian cells is in part controlled by methylation of specific DNA sequences. Carcinogens may act by altering the normal epigenetic controls of gene activity in specialised cells, and thereby produce aberrant heritable phenotypes. It is known that agents which inhibit DNA methylation can be carcinogenic and that tumour cells are altered in DNA methylation.     

DNA甲基化在精子发生中的作用

精子发生(spermatogenesis)是一个高度特化的细胞复杂分化过程,其中DNA二核苷酸CpG甲基化变化与基因转录激活、染色质改构以及遗传印记相关,并且该甲基化与基因表达之间的关系是非直接的,其可通过染色质结构的改变或DNA与蛋白质的相互作用来介导。本文着重介绍精子发生过程中DNA甲基化及其跨代遗传风险、DNA甲基转移酶的调控机制以及DNA甲基化与男性不育之间的关系等,为不育症的防治、精子表观遗传质量评价以及降低辅助生殖技术后代表观遗传疾病风险等提供基础资料。     

Common methods for cytosine methylation analysis in DNA

The review considers the methods most commonly used to detect DNA methylation, their advantages, potential limitations, and selection for various purposes. A detailed protocol is described for bisulfite treatment, which is used as a preliminary step in the majority of DNA methylation assays.     

Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization,T-DNA methylation and conditional expression of opine genes

Summary Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having the auxin locus of the TL-region inactivated by a Tn1831 insertion, were investigated for their T-DNA structure and expression. It has been described previously (28) that in addition to clones with an expected phenotype (phytohormone independent growth in tissue culture (Aut⁺), shoot regeneration (Reg⁺) and octopine synthesis (Ocs⁺)), clones were obtained with an aberrant phenotype. One of these clones, TSO38, is Aut⁺Reg⁺ but shows little or no octopine synthesis activity (Ocs^-). Subclones of TSO38, however, are either Ocs^- or Ocs⁺. Ocs^- shoots become Ocs⁺ under certain states of differentiation, indicating that the octopine synthase gene is present. The fact that in the Ocs^- subclones the octopine synthase gene is not expressed, is probably due to DNA methylation (29). The present paper describes that shoots derived from both an Ocs⁺ and an Ocs^- subclone of TSO38, which were negative for the presence of mannopine (Mas^-) and agropine (Ags^-), became Mas⁺Ags⁺ after culturing on medium containing the hypomethylating agent 5-azacytidine. This means that both in the Ocs^- line and in the Ocs⁺ line expression of TR-DNA opine genes most likely was hampered by DNA methylation. The T-DNA structures of an Ocs^- and an Ocs⁺ TSO38 subclone proved to be identical and surprisingly complex. No intact copy of Tn1831 was present. TL-DNA and TR-DNA segments, present in high copy numbers, were truncated; several T-DNA segments existed in tandem arrangements. When DNA from an Ocs⁺ and an Ocs^- subclone of TSO38 were compared for cleavability by the methylation sensitive restriction enzymes HpaII and MspII, differences were detected, but it became also clear that both lines contained methylated T-DNA segments. This indicates that the Ocs^- and the Ocs⁺ TSO38 subclones differ only quantitatively in respect to degree of T-DNA methylation.     

Silencing of transgene expression in mammalian cells by DNA methylation and histone modifications in gene therapy perspective

DNA methylation and histone modifications are vital in maintaining genomic stability and modulating cellular functions in mammalian cells. These two epigenetic modifications are the most common gene regulatory systems known to spatially control gene expression. Transgene silencing by these two mechanisms is a major challenge to achieving effective gene therapy for many genetic conditions. The implications of transgene silencing caused by epigenetic modifications have been extensively studied and reported in numerous gene delivery studies. This review highlights instances of transgene silencing by DNA methylation and histone modification with specific focus on the role of these two epigenetic effects on the repression of transgene expression in mammalian cells from integrative and non-integrative based gene delivery systems in the context of gene therapy. It also discusses the prospects of achieving an effective and sustained transgene expression for future gene therapy applications.